An aptasensor using ceria electrodeposited-screen-printed carbon electrode for detection of epithelial sodium channel protein as a hypertension biomarker

Abstract

Epithelial sodium channel (ENaC) is a transmembrane protein that has an essential role in maintaining the levels of sodium in blood plasma. A person with a family history of hypertension has a high enough amount of ENaC protein in the kidneys or other organs, so that the ENaC protein acts as a marker that a person is susceptible to hypertension. An aptasensor involves aptamers, which are oligonucleotides that function similar to antibodies, as sensing elements. An electrochemical aptasensor for the detection of ENaC was developed using a screen-printed carbon electrode (SPCE) which was modified by electrodeposition of cerium oxide (CeO₂). The aptamer immobilization was via the streptavidin-biotin system. The measurement of changes in current of the active redox [Fe(CN)₆]^3-/4- was carried out by differential pulse voltammetry. The surfaces of SPCE and SPCE/CeO₂ were characterized using scanning electron microscopy, voltammetry and electrochemical impedance spectroscopy. The Box-Behnken experimental optimization design revealed the streptavidin incubation time, aptamer incubation time and streptavidin concentrations were 30 min, 30 min and 10.8 µg ml^-1, respectively. Various concentrations of ENaC were used to obtain the linearity range of 0.05-3.0 ng ml^-1, and the limits of detection and quantification were 0.012 ng ml^-1 and 0.038 ng ml^-1, respectively. This aptasensor method has the potential to measure the ENaC protein levels in urine samples as well as to be a point-of-care device.

Keywords: aptamer; aptasensor; biosensor; ceria; epithelial sodium channel (ENaC); hypertension.

Figure 1.

Design schematic of the ENaC aptasensor.

Figure 2.

Differential pulse voltammogram of various concentration of cerium nitrate; the response was measured using DPV with a scan rate of 0.05 V s⁻¹ over a potential range of –1.0 to +1.0 V; the optimized cerium concentrations were (a) 25.0, (b) 50.0, (c) 75.0 and (d) 100.0 µg ml⁻¹.

Figure 3.

(a) Microscopic surface morphology for bare SPCE. (b) SEM morphology of SPCE/CeO₂. (c) Differential pulse voltammogram of 10 mM [Fe(CN)₆]^3−/4− in KCl 0.1 M on: a, bare SPCE; b, SPCE/CeO₂; c, SPCE/CeO₂/streptavidin; d, SPCE/CeO₂/streptavidin/aptamer; and e, SPCE/CeO₂/streptavidin/aptamer/ENaC. (d) Nyquist plot of EIS results at a frequency of 100 Hz for: a, bare SPCE; b, SPCE/CeO₂; c, SPCE/CeO₂/streptavidin; and d, SPCE/CeO₂/streptavidin/aptamer.

Figure 4.

(a) The decrease of peak current in voltammograms for determination of each ENaC concentration a–g (0.046875, 0.09375, 0.1875; 0.375, 0.75; 1.5 and 3.0 ng ml⁻¹) using 10 mM of [Fe(CN)₆]^3−/4− in 0.1 M KCl. (b) Plot of current to concentration.